Vascular endothelial growth factor (VEGF) is a potent mediator of enhanced vascular permeability, and published data support a significant role for VEGF in development of the acute respiratory distress syndrome (ARDS). However, VEGF can also stimulate endothelial cell proliferation and chemotaxis, potential reparative responses to vascular injury. Because cellular response to VEGF may be either injurious or reparative, understanding how the endothelium transduces specific VEGF signals is critical. Neuropilin-1 (Npn-1) is an endothelial cell surface receptor that binds VEGF, and can modulate VEGF receptor-2 (VEGFR2) signaling in vitro. Preliminary data suggest Npn-1 is required for VEGF-induced vascular permeability, but is not necessary for VEGF-mediated endothelial chemotaxis. Differential expression or activation of Npn-1 may therefore control the balance between injurious and reparative effects of VEGF on the endothelium. The hypothesis to be tested is that specific endothelial responses to VEGF are determined by activation of distinct receptor complexes, such that Npn-1 selectively regulates VEGF-induced vascular barrier dysfunction. Specific aim 1 will evaluate whether Npn-1 mediates VEGF-induced endothelial permeability in vitro, and if VEGFR2 is required for this response. Studies will take advantage of the unique ligand-receptor specificity of VEGF isoforms and family members, and utilize endothelial cells stably over- expressing Npn-1and/or VEGFR2. In Specific aim 2. transgenic mice will be studied to evaluate whether inducible, endothelial-specific, Npn-1 deletion alters lung vascular barrier function, and if Npn-1inhibition attenuates pulmonary vascular permeability after inducible over-expression of lung VEGF. Finally, Specific aim 3 will utilize inducible, endothelial-specific, Npn-1 knockout mice to evaluate the role of Npn-1 in the development of pulmonary edema in two independent models of acute lung injury. These studies will use a combination of in vitro and in vivo approaches to determine whether VEGF signals through Npn-1to generate pulmonary vascular barrier dysfunction. The proposed experiments represent an important first step in the development of VEGF-targetedtreatment approaches for ARDS. Relevance to public health: Pulmonary edema is the primary symptom of ARDS, a syndrome with 30-40% mortality. Understanding basic mechanisms contributing to the development of pulmonary edema in ARDS may suggest novel therapeutic strategies for managing critically ill patients with this disorder.